RESUMEN

During the periparturient period, both oxidative stress, and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB), which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin-2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for (1) treatment with different concentrations of hydrogen peroxide (H2O2; 0, 25, 50, 100, 200, or 400 µM) for 2 h; (2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 µM H2O2 for 2 h; (3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 mM N-acetylcysteine (NAC) for 2 h; (4) transfection with or without TXN2-overexpressing plasmid for 48 h and then treatment with or without 200 µM H2O2 for 2 h. High concentrations of H2O2 (200 and 400 µM) decreased protein and mRNA abundance of TXN2, reduced total antioxidant capacity (T-AOC) and ATP content in adipocytes. Moreover, 200 and 400 µM H2O2 reduced protein abundance of inhibitor of kappa B α (IκBα), increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α (TNFA) and interleukin-1B (IL-1B), suggesting that H2O2-induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B, decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H2O2-induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H2O2, overexpression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H2O2-induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B, as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response.

Asunto(s)

Adipocitos , Peróxido de Hidrógeno , FN-kappa B , Estrés Oxidativo , Transducción de Señal , Tiorredoxinas , Animales , Bovinos , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Femenino

RESUMEN

Elevated intraocular pressure (IOP) in glaucoma causes retinal ganglion cell (RGC) loss and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. In this paper, we aimed to develop a novel gene therapy for glaucoma using an AAV2-based thioredoxin 2 (Trx2)-exoenzyme C3 transferase (C3) fusion protein expression vector (scAAV2-Trx2-C3). We evaluated the therapeutic effects of this vector in vitro and in vivo using dexamethasone (DEX)-induced glaucoma models. We found that scAAV2-Trx2-C3-treated HeLa cells had significantly reduced GTP-bound active RhoA and increased phosphor-cofilin Ser3 protein expression levels. scAAV2-Trx2-C3 was also shown to inhibit oxidative stress, fibronectin expression, and alpha-SMA expression in DEX-treated HeLa cells. NeuN immunostaining and TUNEL assay in mouse retinal tissues was performed to evaluate its neuroprotective effect upon RGCs, whereas changes in mouse IOP were monitored via rebound tonometer. The present study showed that scAAV2-Trx2-C3 can protect RGCs from degeneration and reduce IOP in a DEX-induced mouse model of glaucoma, while immunohistochemistry revealed that the expression of fibronectin and alpha-SMA was decreased after the transduction of scAAV2-Trx2-C3 in murine eye tissues. Our results suggest that AAV2-Trx2-C3 modulates the outflow resistance of the trabecular meshwork, protects retinal and other ocular tissues from oxidative damage, and may lead to the development of a gene therapeutic for glaucoma.

Asunto(s)

Glaucoma , Presión Intraocular , Humanos , Ratones , Animales , Células Ganglionares de la Retina/metabolismo , Fibronectinas/metabolismo , Tiorredoxinas/metabolismo , Células HeLa , Transferasas/metabolismo , Glaucoma/genética , Glaucoma/terapia , Glaucoma/metabolismo , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: To study the effects of L-carnitine (LC) combined with pancreatic kininogenase on thioredoxin 2 (Trx 2), thioredoxin reductase 1 (TrxR 1), and sperm quality in patients with oligoasthenospermia. METHODS: A total of 300 male infertility patients with oligoasthenospermia who were treated in the andrology clinic of our hospital from December 2019 to December 2020 were randomly divided into an LC group and combined treatment group, and 50 males with normal semen were selected as a control group. The computer-assisted semen analysis system (CASA) was used to detect the total number, vitality, and forward motility of the sperm before and after treatment, and sperm morphology was detected by the Diff-Quik method of the sperm staining kit. Sperm chromatin dispersion (SCD) method was used to detect sperm DNA fragments, and Western-blot was used to detect the protein expression of Trx 2 and TrxR 1. RESULTS: There were no significant differences in sperm density, motility rate, forward motile sperm rate, and DNA fragmentation rate in oligoasthenospermia patients before treatment (P>0.05). However, after 1 month of treatment, the sperm density, motility rate, and forward motile sperm rate were all higher than before treatment (P<0.05), while the DNA fragmentation rate was lower than before treatment. At the same time, each index of semen in the combination group was higher than that in the LC group (P<0.05), and the total effective rate in the combination group was significantly higher than in the LC group (P<0.01). The expression of Trx2 protein in oligoasthenospermia patients was significantly increased (P<0.05), while the expression of TrxR1 protein was significantly decreased (P<0.05). After 3 months of treatment, the expression of Trx2 protein was significantly decreased (P<0.05), while the expression of TrxR1 protein was significantly increased (P<0.05). CONCLUSIONS: The results suggest Trx 2 and TrxR 1 may be candidate protein markers for oligoasthenospermia. LC combined with pancreatic kininogenase in the treatment of male oligoasthenospermia can effectively promote sperm maturation, enhance sperm motility, and improve semen quality, which has high application value.

RESUMEN

BACKGROUND: Poor dietary habits such as an over consumption of high fructose and high fat diet are considered as the major culprit for the induction of diabetes associated liver injury. Diabetes mellitus is a metabolic disorder that affects various vital organs of the body especially the kidney, brain, heart, and liver. The high fructose and high fat (HFHF) diet worsen the metabolic conditions by producing various pathogenic burdens such as oxidative stress, inflammation, etc. on liver. The hyperlipidemic and hyperglycemic conditions induced by HFHF diet leads to the generation of various proinflammatory mediators like TNFα, interleukin and cytokines. AIM AND METHODS: The systematic bibliographical literature survey was done with the help of PubMed, Google scholar and MedLine to identify all pathological and molecular concerened with HFHF induced diabetic liver injury. The consumption of HFHF diet leads to an increase in mitochondrial oxidative stress thereby decreases the liver protective antioxidants required for cell viability. HFHF diet disturbs lipid and lipoprotein clearance by elevating the level of apolipoprotein CIII and impairing the hydrolysis of triglyceride. As a result, there is an increase in free fatty acid concentration, triglycerides and diacylglycerol in the liver which further triggers the situation of insulin resistance. CONCLUSION: The focus of present review is based upon the various pathological, genetic and molecular mechanism involved in the development of high-fat high fructose diet induced diabetic liver injury. However, the current review also documented few shreds of evidence related to various microRNAs (miR-31, miR-33a, miR-34a, miR-144, miR-146b, miR-150) concerned to HFHF diet which play an important role in the pathogenesis of diabetes associated liver injury Dietary life style modification may prove beneficial in the management of various metabolic disorders.

Asunto(s)

Complicaciones de la Diabetes/etiología , Dieta Alta en Grasa , Fructosa/administración & dosificación , Enfermedad del Hígado Graso no Alcohólico/etiología , Microbioma Gastrointestinal , Humanos , Resistencia a la Insulina , Lípidos/sangre , MicroARNs , Estrés Oxidativo

RESUMEN

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.

Asunto(s)

Infecciones Bacterianas/veterinaria , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Smegmamorpha/inmunología , Tiorredoxinas/inmunología , Animales , Infecciones Bacterianas/inmunología , ADN Complementario/genética , Enfermedades de los Peces/microbiología , Proteínas de Peces/inmunología , Regulación de la Expresión Génica , Inmunidad Innata , Filogenia , Smegmamorpha/genética , Tiorredoxinas/genética

RESUMEN

PURPOSE: To investigate the role of thioredoxin 2 (Trx2) inhibition induced by intracellular methylglyoxal (MGO) in pancreatic beta-cell mitochondrial dysfunction and apoptosis. METHODS: Rat pancreatic beta-cell line INS-1 cells were treated with Glo1 siRNAs or exogenous MGO to increase intracellular MGO. AGEs formation was detected by ELISA and mitochondrial ROS was detected by probe MitoSOX. Transmission electron microscopy (TEM) analysis and ATP content were measured to evaluate mitochondrial function. Trx2 expression was manipulated by overexpression with recombinant Trx2 lentivirus or knockdown with Trx2 siRNAs, and effects on apoptosis and insulin secretion were measured by flow cytometry and ELISA, respectively. RESULTS: The increase of intracellular MGO by Glo1 blockage or MGO treatment led to advanced glycation end products (AGEs) overproduction, mitochondrial ROS increase, and insulin secretion paralysis. These were probably due to MGO-induced inhibition of mitochondrial Trx2. Trx2 inhibition by blockage of either Glo1 or Trx2 impaired mitochondrial integrity, inhibited cytochrome C oxidases subunit 1 and 4 (Cox1 and Cox4) expression and further reduced ATP generation, and all of these might lead to insulin paralysis; whereas Trx2 overexpression partially reversed MGO-induced oxidative stress, attenuated insulin secretion by preventing mitochondrial damage. Trx2 overexpression also retarded MGO-induced apoptosis of INS-1 cell through inhibiting ASK1 activation and downregulation of the ASK1-p38 MAPK pathway. CONCLUSIONS: Our results reveal a possible mechanism for beta-cell oxidative damage upon intracellular MGO-induced Trx2 inactivation and mitochondrial dysfunction and apoptosis.

Asunto(s)

Piruvaldehído , Tiorredoxinas , Animales , Apoptosis , Mitocondrias/metabolismo , Estrés Oxidativo , Piruvaldehído/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo

RESUMEN

OBJECTIVE: Sick sinus syndrome (SSS) is associated with loss of HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4) function in the cardiac conduction system. The underlying mechanism for SSS remains elusive. This study is to investigate how mitochondrial oxidative stress induces HCN4 downregulation associated with in sick sinus syndrome. METHODS AND RESULTS: Trx2lox/lox mice were crossed with α-myosin heavy chain (α-Mhc)-Cre and Hcn4-CreERT2 deleter mice to generate Trx2 deletion mice in the whole heart (Trx2cKO) and in the conduction system (Trx2ccsKO), respectively. Echocardiography was applied to measure hemodynamics and heart rhythm. Histological analyses, gene profiling and chromatin immunoprecipitation were performed to define the mechanism by which thioredoxin-2 (Trx2) regulates HCN4 expression and cardiac function. Trx2cKO mice displayed dilated cardiomyopathy, low heart rate, and atrial ventricular block (AVB) phenotypes. Immunofluorescence revealed that HCN4 expression was specifically reduced within the sinoatrial node in Trx2cKO mice. Interestingly, Trx2ccsKO mice displayed low heart rate and AVB without dilated cardiomyopathy. Both mRNA and protein levels of HCN4 were reduced in the sinoatrial node, suggesting transcriptional HCN4 regulation upon Trx2 deletion. ChIP indicated that the binding of MEF2 to the HCN4 enhancer was not altered by Trx2 deletion; however, histone 3 acetylation at the MEF2 binding site was decreased, and expression of histone deacetylase 4 (HDAC4) was elevated following Trx2 deletion. Moreover, HDAC4 binding to the HCN4 enhancer was mediated by MEF2. Mitochondrial ROS were increased by Trx2 deletion and importantly, mitochondria-specific ROS scavenger MitoTEMPO suppressed HDAC4 elevation, HCN4 reduction, and sinus bradycardia in Trx2ccsKO mice. CONCLUSION: In the conduction system, Trx2 is critical for maintaining HCN4-mediated normal heart rate. Loss of Trx2 reduces HCN4 expression via a mitochondrial ROS-HDAC4-MEF2C pathway and subsequently induces sick sinus syndrome in mice.

Asunto(s)

Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Mitocondrias Cardíacas/metabolismo , Estrés Oxidativo , Síndrome del Seno Enfermo/genética , Síndrome del Seno Enfermo/patología , Tiorredoxinas/metabolismo , Animales , Bradicardia/complicaciones , Bradicardia/metabolismo , Bradicardia/patología , Cardiomiopatía Dilatada/complicaciones , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Elementos de Facilitación Genéticos/genética , Histona Desacetilasas/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Factores de Transcripción MEF2/metabolismo , Ratones Noqueados , Modelos Biológicos , Estrés Oxidativo/genética , Fenotipo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Síndrome del Seno Enfermo/complicaciones , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/patología

RESUMEN

OBJECTIVE: Mitochondrial thioredoxin 2 (Trx2) is a vital mitochondrial redox protein that mediates normal protein thiol reduction and provides electrons to peroxiredoxin 3 (Prx3) to scavenge H2O2 in mitochondria. It has been widely reported that Trx2 deletion in cells or mice generates massive reactive oxygen species (ROS) which have been implicated in many pathological processes. On the contrary, how ROS regulate Trx2 processing and activity remains to be elucidated. APPROACH AND RESULTS: Here we show that excess ROS induce endothelial cell senescence concomitant with an attenuation of Trx2 processing in which Trx2 presequence [i.e., mitochondrial targeting signal peptide (MTS)] is cleaved to generate a mature form. Mutation analyses indicate that Trx2 processing is mediated by mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP)-recognition sites within the MTS. Interestingly, a mutation at a SUMO- interacting motif (SIM), but not the catalytic sites within the mature Trx2 protein, completely blocks Trx2 processing with no effect on Trx2 mitochondrial targeting. Consistently, chemical inhibition of protein SUMOylation attenuates, while SUMOylation agonist promotes, Trx2 processing. Moreover, we identify the α-MPP subunit is a SUMOylated protein that potentially mediates Trx2-binding and cleavage. Furthermore, the unprocessed form of Trx2-SIM is unable to protect cells from both ROS generation and oxidative stress-induced cellular senescence. CONCLUSION: Our study reveals that a unique SUMO-interacting motif of Trx2 is critical for its mitochondrial processing and subsequent anti-oxidant/antisenescence activities.

RESUMEN

Thioredoxin system plays an important role in antioxidative stress, thioredoxin 2 (Trx2) being one of the most important components in the thioredoxin system. The full-length cDNA sequence of thioredoxin 2 from Euphausia superba (EsTrx2) is 1276 bp and contain a 5' untranslated region (UTR) of 94 bp, a 3' UTR of 741 bp and an open reading frame (ORF) of 441 bp, encoding a putative protein of 146 amino acids. Multiple sequence alignments have indicated that EsTrx2 possesses a conserved (-Cys-Gly-Pro-Cys-) CGPC redox-active site. EsTrx2 shares 62.3% identity with the swimming crab (Portunus trituberculatus) Trx2. The predicted three-dimensional structure of EsTrx2 consists of a thioredoxin fold. The high similarity and phylogenetic analysis have indicated that EsTrx2 is a member of the mitochondrial Trx2 sub-family. The recombinant EsTrx2 (rEsTrx2) was constructed and expressed in Escherichia coli BL21 (DE3). The rEsTrx2 protein showed high redox activity and antioxidant capacity at temperature from 4 to 37 °C. All results indicated that EsTrx2 was involved in the oxidative stress response of E. superba.

Asunto(s)

Euphausiacea/citología , Euphausiacea/genética , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Animales , Antioxidantes/metabolismo , Secuencia de Bases , Clonación Molecular , Modelos Moleculares , Conformación Proteica , Análisis de Secuencia , Tiorredoxinas/química

RESUMEN

Thioredoxin 2 (Trx2), as a member of the thioredoxin system in mitochondria, is involved in controlling mitochondrial redox state. However, the role of Trx2 in cardiac biology is not fully understood. In the present study, the expression of Trx2 is silenced in quiescent neonatal rat ventricular cardiomyocytes (NRVCs) and mitochondrial respiratory function and cardiomyocyte hypertrophy are assessed. The results show that Trx2 depletion does not induce significant cytotoxicity in quiescent NRVCs. Remarkably, Trx2 depletion results in cardiomyocyte hypertrophy as determined by increased cell size and protein synthesis. Furthermore, Trx2 depletion inhibits AMPK activity and AMPK activator reversed cellular hypertrophy. Trx2 depletion enhances mitochondrial ROS generation without impact on cellular ROS level. Trx2 depletion has no effect on mitochondrial biogenesis. Specifically, Trx2 depletion increases mitochondrial respiration flux and total ATP concentration under quiescent conditions. To decipher the relationship between ROS generation, mitochondrial respiration flux, and AMPK signaling, mitochondrial metabolism and ROS was specifically inhibited, and the results show that AMPK inactivation and hypertrophic response in Trx2-silenced cells is reversed by respiration blockers but not ROS scavenger. In conclusion, these results show that beyond mitochondrial ROS scavenging, Trx2 controls mitochondrial respiratory function in quiescent cardiomyocytes and is implicated in cardiomyocyte hypertrophy via AMPK signaling.

Asunto(s)

Proteínas Quinasas Activadas por AMP/genética , Adenosina Trifosfato/metabolismo , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Tamaño de la Célula , Regulación de la Expresión Génica , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Oligomicinas/farmacología , Oxidación-Reducción/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ribonucleótidos/farmacología , Rotenona/farmacología , Transducción de Señal , Tiorredoxinas/antagonistas & inhibidores , Tiorredoxinas/metabolismo

RESUMEN

Mitochondrial energy metabolism depends upon high-flux and low-flux electron transfer pathways. The former provide the energy to support chemiosmotic coupling for oxidative phosphorylation. The latter provide mechanisms for signaling and control of mitochondrial functions. Few practical methods are available to measure rates of individual mitochondrial electron transfer reactions; however, a number of approaches are available to measure steady-state redox potentials (E h) of donor/acceptor couples, and these can be used to gain insight into rate controlling reactions as well as mitochondrial bioenergetics. Redox changes within the respiratory electron transfer pathway are quantified by optical spectroscopy and measurement of changes in autofluorescence. Low-flux pathways involving thiol/disulfide redox couples are measured by redox Western blot and mass spectrometry-based redox proteomics. Together, the approaches provide the opportunity to develop integrated systems biology descriptions of mitochondrial redox signaling and control mechanisms.

Asunto(s)

Metabolismo Energético , Espectrometría de Masas/métodos , Mitocondrias/metabolismo , Proteómica/métodos , Western Blotting/instrumentación , Western Blotting/métodos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Cisteína/metabolismo , Transporte de Electrón , Glutatión/metabolismo , Células HT29 , Humanos , Espectrometría de Masas/instrumentación , Oxidación-Reducción , Proteómica/instrumentación , S-Nitrosotioles/metabolismo

RESUMEN

BACKGROUND: Human babesiosis is an infectious disease that is epidemic in various regions all over the world. The predominant causative pathogen of this disease is the intra-erythrocytic parasite Babesia microti. The thioredoxin system is one of the major weapons that is used in the resistance to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host immune system. In other intra-erythrocytic apicomplexans like the malaria parasite Plasmodium falciparum, anti-oxidative proteins are promising targets for the development of anti-parasitic drugs. However, to date, the sequences and biological properties of thioredoxins and thioredoxin-like molecules of B. microti remain unknown. Understanding the molecular characterization and function of B. microti thioredoxins may help to develop anti-Babesia drugs and controlling babesiosis. METHODS: The full-length B. microti thioredoxin 2 (BmTrx2) gene was obtained using a rapid amplification of cDNA ends (RACE) method, and the deduced BmTrx2 amino acid sequence was analyzed using regular bioinformatics tools. Recombinant BmTrx2 protein was expressed in vitro and purified using His-tag protein affinity chromatography resins. Reverse transcription PCR, quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx2. Indirect immunofluorescence assay was used to localize BmTrx2 in B. microti. Bovine insulin reduction assays were used to determine the enzyme activity of the purified recombinant BmTrx2 protein. RESULTS: The full-length BmTrx2 was 564 bp with a 408 bp open reading frame encoding a protein of 135 amino acids. The predicted molecular weight of the protein was 15.5 kDa. A conserved thioredoxin-like family domain was found in BmTrx2. The expression of BmTrx2 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. The results of Western blot analysis showed the native BmTrx2 in parasite lysates could be detected by mouse anti-BmTrx2 serum and that the recombinant BmTrx2 protein could be recognized by serum of B. microti-infected mice. Immunofluorescence microscopy showed that BmTrx2 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the purified recombinant BmTrx2 protein possesses antioxidant enzyme activity. Dihydroartemisinin and quinine, known anti-malaria drugs, and clindamycin, a known anti-babesiosis drug, induced significantly higher upregulation of BmTrx2 mRNA. CONCLUSIONS: Our results indicate that BmTrx2 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs.

Asunto(s)

Antiprotozoarios/farmacología , Babesia microti/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Tiorredoxinas/biosíntesis , Tiorredoxinas/genética , Animales , Western Blotting , Bovinos , Biología Computacional , Técnica del Anticuerpo Fluorescente Indirecta , Perfilación de la Expresión Génica , Insulina/metabolismo , Ratones , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Tiorredoxinas/análisis

RESUMEN

Objective:To explore whether thioredoin-2 (Trx-2) is involved in the development of cataract and to study the effect of Trx-2 on hydrogen peroxide (H2O2)-induced injury in human lens epithelial cells.Methods:A total of 10 volunteers (removing the lens due totraumatism) and 30 patients received phacoemulsification (age more than 60 years) were selected.The expression of Trx-2 protein in lens epithelial cells from cataract patients and volunteers were detected by the immunohistochemical streptavidin-peroxidase (SP) method.SRA01/04 cells were cultured and were divided into six groups according to different treatment:a control group,H2O2-treated groups at 20,50 or 100 μmol/L,a negative control group (transfected with pCMV6 plasmid plus 100 μmol/L H2O2),and a Trx-2 overexpression group (transfected with pCMV6-Trx-2 plasmid plus 100 μmol/L H2O2).Methyl thiazolyltetrazolium (MTT) assay and flow cytometry was performed to measure the cell viability and apoptosis for SRA01/04 cells,respectively.The activities of superoxide dismutase (SOD) and catalase (CAT),the content of glutathione (GSH) and malondialdehyde (MDA) in human lens epithelial cells were measured via chemical chromatometry.Western blot was used to measure the protein levels of Trx-2,B-cell lymphoma 2 protein (Bcl-2),Bcl-2 associated X protein (Bax) and caspase-3.Results:Compared with the volunteers,the expression of Trx-2 was significantly decreased in lens epithelial cells in patients with cataract (P＜0.05).Compared with the control group,the expression of Trx-2 protein in the 20,50 or 100 μmol/L H2O2 groups was decreased (all P＜0.05).Compared with the control group,the cell survival rates were decreased in the 100 μmol/L H2O2 group and the negative control group (both P＜0.05),along with enhanced apoptotic rates,inhibited cellular SOD activities and CAT activities,reduced GSH contents,augmented MDA contents,down-regulated Trx-2 and Bcl-2 expression and up-regulated Bax and caspase-3 expression (all P＜0.05).Compared with the negative control group,the cell survival rate was increased in the Trx-2 overexpression group (P＜0.05),along with suppressed apoptosis,increased SOD activities and CAT activities,elevated GSH contents,decreased MDA content,up-regulated Trx-2 and Bcl-2 expression and down-regulated Bax and caspase-3 expression (P＜0.05).Conclusion:Trx-2 might be involved in the apoptosis of lens epithelial cells in patients with cataract.The overexpression of Trx-2 obviously attenuated H2O2-induced injury of human lens epithelial cells,which might be associated with the inhibition of H2O2-mediated oxidative stress.

RESUMEN

Mitochondrial oxidative stress is thought to be a key contributor towards the development of diabetic cardiomyopathy. Thioredoxin 2 (Trx2) is a mitochondrial antioxidant that, along with Trx reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), scavenges H2O2 and offers protection against oxidative stress. Our previous study showed that TrxR inhibitors resulted in Trx2 oxidation and increased ROS emission from mitochondria. In the present study, we observed that TrxR inhibition also impaired the contractile function of isolated heart. Our studies showed a decrease in the expression of Trx2 in the high glucose-treated H9c2 cardiac cells and myocardium of streptozotocin (STZ)-induced diabetic rats. Overexpression of Trx2 could significantly diminish high glucose-induced mitochondrial oxidative damage and improved ATP production in cultured H9c2 cells. Notably, Trx2 overexpression could suppress high glucose-induced atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) gene expression. Our studies suggest that high glucose-induced mitochondrial oxidative damage can be prevented by elevating Trx2 levels, thereby providing extensive protection to the diabetic heart.

Asunto(s)

Cardiomiopatía Hipertrófica/etiología , Cardiomiopatía Hipertrófica/metabolismo , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Tiorredoxinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Expresión Génica , Humanos , Hiperglucemia/sangre , Masculino , Ratones , Mitocondrias/genética , Contracción Miocárdica/genética , Oxidación-Reducción , Ratas , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/genética , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/metabolismo

RESUMEN

Thioredoxin 2 (Trx2) is a pivotal mitochondrial protein that regulates redox signaling. The mitochondrial Trx2 is expressed ubiquitously, but it is found at the highest levels in metabolically active tissues like the heart. Global gene knockout of Trx2 results in embryonic lethality, likely due to the increased cellular oxidative stress. Moreover, mice with cardiac-specific Trx2 deletion develop spontaneous dilated cardiomyopathy (DCM), correlating with increased apoptosis stress kinase-1 (ASK1) signaling and increased cardiomyocyte apoptosis. Cardiomyocyte apoptosis is a common mechanism in the pathogenesis of heart failure. Our results show that Trx2 is essential for maintaining cardiac function. In this chapter, we summarize the key mechanistic role of Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species (ROS) generation and by inhibiting ASK1-dependent apoptosis in heart failure. Trx2 and ASK1 represent promising targets to develop therapeutic strategies for the treatment of DCM and heart failure.

Asunto(s)

Insuficiencia Cardíaca/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal , Tiorredoxinas/metabolismo , Animales , Apoptosis , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Mitocondrias Cardíacas/patología , Contracción Miocárdica , Miocitos Cardíacos/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Preconditioning by brief ischemic episode induces tolerance to a subsequent lethal ischemic insult, and it has been suggested that reactive oxygen species are involved in this phenomenon. Thioredoxin 2 (Trx2), a small protein with redox-regulating function, shows cytoprotective roles against oxidative stress. Here, we had focused on the role of Trx2 in ischemic preconditioning (IPC)-mediated neuroprotection against oxidative stress followed by a subsequent lethal transient cerebral ischemia. Animals used in this study were randomly assigned to six groups; sham-operated group, ischemia-operated group, IPC plus (+) sham-operated group, IPC + ischemia-operated group, IPC + auranofin (a TrxR2 inhibitor) + sham-operated group and IPC + auranofin + ischemia-operated group. IPC was subjected to a 2 minutes of sublethal transient ischemia 1 day prior to a 5 minutes of lethal transient ischemia. A significant loss of neurons was found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-group 5 days after ischemia-reperfusion; in the IPC + ischemia-operated-group, pyramidal neurons in the SP were well protected. In the IPC + ischemia-operated-group, Trx2 and TrxR2 immunoreactivities in the SP and its protein level in the CA1 were not significantly changed compared with those in the sham-operated-group after ischemia-reperfusion. In addition, superoxide dismutase 2 (SOD2) expression, superoxide anion radical ( O2-) production, denatured cytochrome c expression and TUNEL-positive cells in the IPC + ischemia-operated-group were similar to those in the sham-operated-group. Conversely, the treatment of auranofin to the IPC + ischemia-operated-group significantly increased cell damage/death and abolished the IPC-induced effect on Trx2 and TrxR2 expressions. Furthermore, the inhibition of Trx2R nearly cancelled the beneficial effects of IPC on SOD2 expression, O2- production, denatured cytochrome c expression and TUNEL-positive cells. In brief, this study shows that IPC conferred neuroprotection against ischemic injury by maintaining Trx2 and suggests that the maintenance or enhancement of Trx2 expression by IPC may be a legitimate strategy for therapeutic intervention of cerebral ischemia.

Asunto(s)

Isquemia Encefálica/metabolismo , Región CA1 Hipocampal/metabolismo , Precondicionamiento Isquémico , Neuronas/metabolismo , Neuroprotección/fisiología , Tiorredoxinas/metabolismo , Animales , Auranofina/farmacología , Isquemia Encefálica/patología , Isquemia Encefálica/prevención & control , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Citocromos c/metabolismo , Inhibidores Enzimáticos/farmacología , Gerbillinae , Precondicionamiento Isquémico/métodos , Masculino , Neuronas/efectos de los fármacos , Neuronas/patología , Neuroprotección/efectos de los fármacos , Estrés Oxidativo/fisiología , Distribución Aleatoria , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Tiorredoxina Reductasa 2/antagonistas & inhibidores , Tiorredoxina Reductasa 2/metabolismo

Asunto(s)

Caspasa 3/metabolismo , Polaridad Celular , Microglía/citología , Microglía/enzimología , Animales , Apoptosis , Humanos , Modelos Biológicos

RESUMEN

Age-associated degeneration in the central auditory system, which is defined as central presbycusis, can impair sound localization and speech perception. Research has shown that oxidative stress plays a central role in the pathological process of central presbycusis. Thioredoxin 2 (Trx2), one member of thioredoxin family, plays a key role in regulating the homeostasis of cellular reactive oxygen species and anti-apoptosis. The purpose of this study was to explore the association between Trx2 and the phenotype of central presbycusis using a mimetic aging animal model induced by long-term exposure to d-galactose (d-Gal). We also explored changes in thioredoxin-interacting protein (TXNIP), apoptosis signal regulating kinase 1 (ASK1) and phosphorylated ASK1 (p-ASK1) expression, as well as the Trx2-TXNIP/Trx2-ASK1 binding complex in the auditory cortex of mimetic aging rats. Our results demonstrate that, compared with control groups, the levels of Trx2 and Trx2-ASK1 binding complex were significantly reduced, whereas TXNIP, ASK1 p-ASK1 expression, and Trx2-TXNIP binding complex were significantly increased in the auditory cortex of the mimetic aging groups. Our results indicated that changes in Trx2 and the TXNIP-Trx2-ASK1 signal pathway may participate in the pathogenesis of central presbycusis.

Asunto(s)

Corteza Auditiva/metabolismo , Proteínas Portadoras/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Tiorredoxinas/metabolismo , Envejecimiento , Animales , Antioxidantes/metabolismo , Apoptosis/genética , Corteza Auditiva/citología , Corteza Auditiva/fisiología , Corteza Auditiva/ultraestructura , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Regulación de la Expresión Génica , MAP Quinasa Quinasa Quinasa 5/genética , Malondialdehído/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Tiorredoxinas/genética

RESUMEN

Alcohol abuse suppresses the immune responses of alveolar macrophages (AMs) and increases the risk of a respiratory infection via chronic oxidative stress and depletion of critical antioxidants within alveolar cells and the alveolar lining fluid. Although alcohol-induced mitochondrial oxidative stress has been demonstrated, the oxidation of the mitochondrial thioredoxin redox circuit in response to alcohol has not been examined. In vitro ethanol exposure of a mouse AM cell line and AMs from ethanol-fed mice demonstrated NADPH depletion concomitant with oxidation of mitochondrial glutathione and oxidation of the thioredoxin redox circuit system including thioredoxin 2 (Trx2) and thioredoxin 2 reductase (Trx2R). Mitochondrial peroxiredoxins (Prdx's), which are critical for the reduction of the thioredoxin circuit, were irreversibly hyperoxidized to an inactive form. Ethanol also decreased the mRNAs for Trx2, Trx2R, Prdx3, and Prdx5 plus the mitochondrial thiol-disulfide proteins glutaredoxin 2, glutathione reductase, and glutathione peroxidase 2. Thus, the mitochondrial thioredoxin circuit was highly oxidized by ethanol, thereby compromising the mitochondrial antioxidant capacity and ability to detoxify mitochondrial reactive oxygen species. Oxidation of the mitochondrial thioredoxin redox circuit would further compromise the transient oxidation of thiol groups within specific proteins, the basis of redox signaling, and the processes by which cells respond to oxidants. Impaired mitochondria can then jeopardize cellular function of AMs, such as phagocytosis, which may explain the increased risk of respiratory infection in subjects with an alcohol use disorder.

Asunto(s)

Etanol/farmacología , Macrófagos Alveolares/inmunología , Mitocondrias/patología , Fagocitosis/efectos de los fármacos , Consumo de Bebidas Alcohólicas , Animales , Línea Celular , Depresores del Sistema Nervioso Central/administración & dosificación , Depresores del Sistema Nervioso Central/farmacología , Etanol/administración & dosificación , Glutarredoxinas/biosíntesis , Glutatión/metabolismo , Glutatión Peroxidasa , Glutatión Reductasa/biosíntesis , Ratones , Ratones Endogámicos C57BL , NADP/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo , Peroxirredoxinas/metabolismo , Fagocitosis/inmunología , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo

RESUMEN

Protein export is considered an essential feature of malaria parasite blood stage development. Here, we examined five components of the candidate Plasmodium translocon of exported proteins (PTEX), a complex thought to mediate protein export across the parasitophorous vacuole membrane into the host cell. Using the murine malaria model parasite Plasmodium berghei, we succeeded in generating parasite lines lacking PTEX88 and thioredoxin 2 (TRX2). Repeated attempts to delete the remaining three translocon components failed, suggesting essential functions for EXP2, PTEX150, and heat shock protein 101 (HSP101) during blood stage development. To analyze blood infections of the null-mutants, we established a flow cytometry-assisted intravital competition assay using three novel high fluorescent lines (Bergreen, Beryellow, and Berred). Although blood stage development of parasites lacking TRX2 was affected, the deficit was much more striking in PTEX88 null-mutants. The multiplication rate of PTEX88-deficient parasites was strongly reduced resulting in out-competition by wild-type parasites. Endogenous tagging revealed that TRX2::tag resides in distinct punctate organelles of unknown identity. PTEX88::tag shows a diffuse intraparasitic pattern in blood stage parasites. In trophozoites, PTEX88::tag also localized to previously unrecognized extensions reaching from the parasite surface into the erythrocyte cytoplasm. Together, our results indicate auxiliary roles for TRX2 and PTEX88 and central roles for EXP2, PTEX150, and HSP101 during P. berghei blood infection.

Asunto(s)

Sangre/parasitología , Proteínas de Unión al Calcio/metabolismo , Regulación de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Plasmodium berghei/crecimiento & desarrollo , Proteínas Protozoarias/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Eliminación de Gen , Genes Esenciales , Genes Protozoarios , Prueba de Complementación Genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/genética , Transporte de Proteínas , Proteínas Protozoarias/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Péptidos/genética